Peptides are short strings of covalently bound amino acids (i.e., 2-50 residues) that serve diverse biological roles as hormones, neurotransmitters, immunogens, and more. The functional versatility of these biomolecules has been increasingly leveraged for therapeutic and diagnostic applications. Despite unfavorable properties, including limitations in gastrointestinal absorption, membrane permeability, and susceptibility to degradation, peptides’ high target affinity, specificity, and safety often outperform other therapeutic modalities, such as small molecule or protein drugs.1 Moreover, advances in chemical synthesis strategies incorporating unnatural amino acids and adopting peptide cyclization, stapling, and conjugation have helped improve their potential for various biomedical applications.2 This listicle explores innovative applications of peptides as therapeutics, in building biomaterials for regenerative medicine, and as drug-delivery tools.
Protein aggregation is a biological process often present in neurodegenerative conditions such as Alzheimer’s, Parkinson’s, and Huntington’s disease. In these conditions, protein misfolding and reduced protein stability converge to form insoluble protein aggregates, also known as amyloid fibrils.3 Despite its ties to pathophysiology, a new strategy aims to harness amyloid formation to specifically target and eliminate proteins for therapeutic purposes.
Pept-ins are short peptides with amyloidogenic properties that are designed to induce targeted protein aggregation. Sequence selection for Pept-in synthesis exploits aggregation-prone regions (APRs) commonly found within the hydrophobic core or on surface-exposed protein domains. These APRs generally consist of 5 to 15 residues that can engage in β-strand interactions with homologous protein sequences.3
Therefore, Pept-ins with sequence homology to APRs in a target protein of interest can serve as “amyloid seeds,” promoting and accelerating protein aggregation and reducing its function.3 To achieve the best aggregating activity, Pept-ins are designed as APR tandem repeats. Moreover, select charged amino acids border the APRs to help reduce peptide self-aggregation.
Several Pept-ins have been designed to target tumor, viral, or bacterial proteins.3 For instance, the Pept-in “vascin” was designed to target an amyloidogenic sequence in VEGFR2, a factor critical for the progression of malignant melanoma.4,5 In a VEGFR2-sensitive subcutaneous B16 melanoma syngenic tumor mouse model, vascin reduced the receptor’s activity and concomitantly tumor growth.4 Additionally, a Pept-in designed to specifically target the influenza A viral protein Polymerase basic 2 (PB2) efficiently reduced viral load in the lungs of infected mice.6
Influenza A Polymerase basic 2 (PB2) protein targeting Pept-in design. The Pept-in was designed as tandem repeats of surface-exposed APR sequences from Influenza A PB2 protein. APR tandem repeats are linked by a dipeptide of glycine-serine or proline-proline arginine or aspartic acid and flanked by charged amino acids (e.g., arginine or aspartic acid). Retrieved from Michiels et al. 2020 with modifications: only panels a and b from Figure 1 are shown.6 http://creativecommons.org/licenses/by/4.0/h.
Lastly, APRs from bacterial proteins have also been leveraged to directly kill bacteria or, more recently, improve antibiotics' efficacy.7,8 To kill bacteria, Pept-ins were designed based on APR sequences that showed redundancy throughout the E. coli proteome. Such Pept-ins induced widespread bacterial protein aggregation and effectively reduced bacterial load in vivo in a mouse model of E. coli bladder infection.7
In their most recent application, Pept-ins provide a new approach to extend the medical use of Beta-lactam antibiotics.8 Beta-lactams bind and inhibit bacterial enzymes involved in peptidoglycan crosslinking, thereby affecting bacterial cell wall formation. However, the rise of bacterial resistance to these agents has significantly reduced their bactericidal effectiveness. Among various resistance strategies, Beta-lactamases, which hydrolyze Beta-lactams, have evolved as a primary protective mechanism in bacteria.
Beta-Lactamase Pept-In design. Synthetic amyloid peptides designed to target extended-spectrum Beta-lactamases, such as TEM-1 and SHV-11 consisted of tandem APR sequence repeats (~7 residues) and incorporated flanking arginine residues and a proline residue as a linker. Adapted from https://switchlab.org/technology/short-stretch-hypothesis-protein-aggregation/
Pept-ins designed to target extended-spectrum Beta-lactamases, such as TEM-1 and/or SHV-11, demonstrated specificity, enhancing the effectiveness of Beta-lactams (i.e., Penicillin G) only against E. coli-resistant strains expressing the matching enzymes. Moreover, Pept-ins improved the outcome of Beta-lactam treatment in vivo in a mouse model of urinary tract infection, significantly reducing bacterial load without toxicity and outperforming the benefits of the clinically used small molecule beta-lactamase inhibitor, tazobactam.8
Hydrogels are soft materials made of tri-dimensionally arranged polymer networks that retain high water content. Their physicochemical properties and, therefore, their utility for biomedical applications are largely dictated by their constituent materials. Hydrogels can be made from natural (e.g., Collagen, Gelatin, Hyaluronic acid) or synthetic (e.g., PEG, PVA, and PCL) basic components. Those based on natural building blocks are more biocompatible and biodegradable and thus provide more opportunities for biomedical applications, such as drug delivery, immunoengineering, tissue engineering, diagnostics, and more.9,10
Synthetic peptides have emerged as attractive biomaterials that can self-assemble through hydrogen bonds and electrostatic interactions to form versatile hydrogels.9 Because the resulting nanostructure of peptide hydrogels resembles the extracellular matrix arrangement, these materials provide ample opportunities as cellular scaffolds. Peptides can also be designed to harbor and present biologically relevant signals or binding sites to promote specific cellular responses, such as proliferation and differentiation. Moreover, current advanced synthetic technologies make it possible to finetune these materials through unique peptide designs to achieve highly functional hydrogels supporting various applications.11 Recently, hydrogels have been functionalized with peptides to support bone tissue engineering applications.
Hydrogel biomaterials provide an effective solution in tissue regeneration approaches, helping transferred stem cells to thrive, as these materials can be highly customized to suit cellular survival and differentiation needs. For bone regeneration, synthetic hydrogels functionalized with peptides must meet unique biological and mechanical properties. As such, materials equipped with biological cues (e.g., growth factors and adhesion molecules) and physical properties (e.g., biodegradability) are highly sought to promote osteogenic differentiation. To this end, a recently developed PEG-based hydrogel was biofunctionalized by incorporating a new biomimetic peptide consisting of a cyclic RGD cell adhesive motif (cRGD) and a BMP-2-derived peptide (DWIVA or cDWIVA).12 Additionally, protease-degradable peptides crosslinking the PEG network were incorporated to achieve desirable mechanical characteristics. 12
Biofunctionalized PEG hydrogel for bone regeneration. PEG-4Mal was modified with the biomimetic peptides and mixed with human mesenchymal stem cells, followed up by cross-linking with a mixture of PEG-diSH and VPM peptide to form cell-loaded biodegradable hydrogels. Retrieved from Oliver-Cervello et al. 2023. Only panel A from Figure 1 is shown. 12 https://creativecommons.org/licenses/by/4.0/
Mesenchymal cells seeded within the new biofunctionalized PEG hydrogel, as opposed to those in a non-functionalized PEG, showed improved morphology indicative of differentiation towards an osteogenic lineage. In agreement with morphological changes, osteogenic differentiation markers were significantly overexpressed by cells within the biofunctionalized PEG hydrogel. Moreover, including biomimetic peptides promoted the expression of metalloproteinases able to break down the VPM peptides crosslinking the PEG network, consequently stimulating desirable cellular morphology.12 Therefore, biochemical and physical optimization of synthetic materials, such as PEG, to emulate the bone extracellular matrix is making possible new opportunities to improve cell-based therapies for bone remodeling.
Q-Peptide functionalized hydrogel for wound healing. Fibroblasts play a critical role in wound repair through deposition and remodeling of extracellular matrix components. Human dermal fibroblasts seeded into a collagen-chitosan hydrogel, functionalized by conjugation to an integrin-binding Q-peptide, increased their expression of various wound healing-related genes and showed unique secretion of anti-fibrotic, and anti-inflammatory cytokines. Retrieved without modifications from Vizely et al. 2023.15 http://creativecommons.org/licenses/by/4.0/
Beyond bone remodeling, hydrogels biofunctionalized with peptides are promising to significantly advance wound healing. Several in vivo studies have demonstrated the efficacy of a collagen-chitosan hydrogel functionalized with an integrin-binding prosurvival peptide or Q-peptide, derived from angiopoietin-1, in accelerating wound closure. By providing stable wet healing conditions and cues that promote keratinocyte migration, survival, favorable fibroblast properties, and immune mobilization, the Q-peptide hydrogel accelerates wound repair and has been deemed as a candidate with clinical promise.13, 14, 15Peptides have lower immunogenicity and toxicity risks, as opposed to proteins, such as antibodies, making them versatile molecules that can be optimized for deep tissue penetration and efficient cellular internalization. Their programmable specificity for cell surface targets makes them ideal for delivering various cargoes (e.g., nanoparticles, and exosomes) to tumor tissues. Once homing-peptide-decorated cargoes reach tumors, they can interact specifically with overexpressed surface proteins and become internalized by tumor cells, thereby improving the efficacy and safety of delivered therapeutics.16
| Tumor-Homing Peptide | Sequence | Target | Cancer Type |
|---|---|---|---|
| RGD4C | ACDCRGDCFCG | Integrin αvβ3 | Melanoma, colon tumor, ovarian tumor glioblastoma |
| iRGD | CRGDR/KGPDC | Integrin αvβ3 | Glioblastoma, melanoma |
| LyP-1 | CGQKRTRGC | P32 | Melanoma |
| K237 | HTMYYHHYQHHL | VEGFR-2 | Breast tumor |
| IL4RPep-1 | CRKRLDRNC | IL4R | Lung tumor, breast tumor, colon tumor |
| mUNO | CSPGAK | CD206 | Breast tumor |
Adapted from Vadevoo et al. 2023. http://creativecommons.org/licenses/by/4.0/
However, tumors in the brain, such as gliomas, present additional delivery barriers. Therapeutic nanocarriers must first traverse the blood–brain barrier (BBB), restricting brain access to most small and large-molecule drugs. Additionally, in gliomas, drugs must be delivered across the blood-brain-tumor-barrier (BBTB), a secondary barrier of neovasculature and specialized endothelial cells surrounding the tumor.17 Therefore, cell-penetrating peptides (CPPs), consisting of short amino acid sequences (i.e., fewer than 30 aa) and having the capacity to traverse biological membranes provide new opportunities for targeted drug delivery to the brain.18
CPPs produced through solid-phase synthesis can be cationic, amphipathic, or hydrophobic to suit the specific application. For example, the positively charged guanidino groups in Arginine residues are critical to establish cell membrane interactions and for internalization either through direct translocation or endocytosis.19 Additionally, inclusion of non-natural amino acids makes it possible to develop CPPs with desirable structural properties, ensuring function and stability. 19
Liposomes can be functionalized with a combination of peptides to achieve efficient drug delivery.18,20 Previously, functionalization with the nicotinic acetylcholine receptor binding peptide CDX and the tumor-homing peptide cyclic RGD were shown to improve the delivery of intact liposomes across the BBB and into glioma cells.21 Functionalization with the CPP Kaposi fibroblast growth factor (kFGF) and a Transferrin receptor binding peptide, able to cross the BBB via receptor-mediated transcytosis, improved liposome delivery to neurons in vivo.22 Significantly, in a similar approach, the dual-functionalization of liposomes with a transferrin peptide and the cell-penetrating peptide Penetratin (Pen) improved the delivery of doxorubicin and erlotinib, resulting in brain tumor regression and increased survival.23
Dual-functionalization of liposomes for drug delivery to glioblastomas. Liposomes decorated with Transferrin peptides facilitate BBB crossing through Transferrin receptor-mediated transcytosis. Additionally, functionalization with penetration enhancer peptides, such as Penetratin, further improves translocation. Retrieved without modification from Mukhtar et al. 2020. 24 https://creativecommons.org/licenses/by/4.0/
More recently, liposomes functionalized with a neurofilament-derived peptide were efficiently internalized by glioblastoma cells in an in vitro BBB and BBTB model. The new peptide consisting of the neurofilament low subunit-tubulin binding site 40–63, NFL-TBS.40–63 peptide (YSSYSAPVSSSLSVRRSYSSSSGS), improved the capacity of liposomes to cross the BBB and BBTB to become preferentially internalized by glioma cells over cancer endothelial cells.25 Although the effectiveness of this NFL peptide remains to be validated in vivo, the in vitro findings are encouragingly supporting its potential for improving drug delivery to gliomas.
